The present invention is a family of eukaryotic expression plasmids useful for gene therapy, genetic immunization or interferon therapy. Such molecules and methods for use are useful in biotechnology, gene therapy, cancer and agriculture.
With the invention in mind, a search of the prior art was conducted. DNA vaccines (genetic vaccines) are a potential disruptive technology, that offer the promise of a new way to immunize humans (or animals) with materials that are entirely gene-based and expressed by the organism's own cells, making an ideal mimic of intracellular antigens.
Methods to improve immune responses to DNA vaccine plasmids are described in the art. For example, the efficacy of a DNA vaccine can be further improved, or tailored for systemic or mucosal immunity, or cancer, allergy, bacterial, intracellular parasite or viral targets, by: coimmunization with costimulatory plasmids (e.g. IL12) to modulate the type of response (TH1 versus TH2 bias); cell death inhibitors or enhancers; or optimization of delivery (e.g. electroporation versus gene gun). Some such methods and molecules are described in, Lemieux, P. 2002 Expert Rev. Vaccines 1: 85-93; Toka F N, Pack C D, Rouse B T. 2004 Immunological reviews 199: 100-112; and Gurunathan S, Klinman D M, Seder R A. 2000 Annu. Rev. Immunol. 18: 927-974, and are included herein by reference. DNA vaccination could also involve utilizing different delivery systems in the prime and the boost, as taught by Buchan S, Gronevik E. Mathiesen I, King C, Stevenson F K, Rice J. 2005 Immunol. 174: 6292-6298 or different injection sites, as taught by Pavlakis G N, Gragerov A, Felber B K. 2004 US Patent Application US20040241140.
DNA vaccination is ideal for rapid deployment vaccines since development times for DNA vaccines are significantly shorter than those for protein or viral vector systems.
Current Obstacles
Regulatory:
Regulatory issues for use of plasmid DNA in humans have been addressed in several recent World Heath Organization (WHO), US Food and Drug Administration (FDA), or European Agency for the Evaluation of Medicinal Products (EMEA) regulatory draft guidances. A key issue is that antibiotic resistance markers, typically kanamycin resistance, are the most commonly utilized selectable markers. The EMEA guidance [European Medicines Agency, 2001. Note for guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products. CPMP/BWP/3088/99] with regard to DNA vaccine plasmids states: “The use of certain selection markers, such as resistance to antibiotics, which may adversely impact on other clinical therapies in the target population,” and “Consideration should be given to avoiding their use, where feasible”. Alternative selection strategies to address this concern are needed.
Efficacy:
Protective immunity in humans and other primates has not been broadly obtained using DNA only vaccination. Primate efficacy has been obtained utilizing DNA vaccines in combination with heterologous protein, inactivated organism, or viral vector boosting. Enhanced immune responses have also been reported when plasmid DNA and purified protein (corresponding to the protein encoded in the plasmid) (Dalemans W., Van Mechelen M V, Bruck C, Friede M. 2003 U.S. Pat. No. 6,500,432; Carrera S D, Grillo J M, de Leon L A L P, Lasa A M, Feyt R P, Rodriguez A V, Obregon J C A, Rivero N A Donato G M 2004 US Patent Application US20040234543), or inactivated virus, (Rangarajan P N, Srinivasan V A, Biswas L, Reddy G S. 2004 US Patent Application US20040096462) are mixed and coinjected.
However, using plasmids in combination with inactivated organisms, proteins or viral vectors in a vaccine (either as a mixture, or sequentially in a prime boost) eliminates most of the benefits of DNA vaccination, including improved safety, reduced cost, and rapid deployment.
DNA vaccines may be incrementally improved by the following methodologies:
Antigen expression: The art teaches that one of the limitations of DNA vaccination is that antigen expression is generally very low. Vector modifications that improve antigen expression (e.g. codon optimization of the gene, inclusion of an intron, use of the strong constitutive cytomegalovirus therein CMV) or CAGG promoters versus weaker or cell line specific promoter) are highly correlative with improved immune responses (reviewed in Manoj S, Babiuk L A, Drunen S V, en Hurk L V. 2004 Critical Rev Clin Lab Sci 41: 1-39). A hybrid CMV promoter (CMV/R), which increased antigen expression, also improved cellular immune responses to HIV DNA vaccines in mice and nonhuman primates (Barouch D H, Yang Z Y, Kong W P, Korioth-Schmitz B, Sumida S M, Truitt D M, Kishko M G, Arthur J C, Miura A, Mascola J R, Letvin N L, Nabel G J. 2005 J. Virol. 79: 8828-8834). A plasmid containing the woodchuck hepatitis virus posttranscriptional regulatory element (a 600 by element that increases stability and extranuclear transport of RNA resulting in enhanced levels of mRNA for translation) enhanced antigen expression and protective immunity to influenza hemagglutinin (HA) in mice (Garg S, Oran A E, Hon H, Jacob J. 2002 J Immunol. 173: 550-558). These studies teach that improvement in expression beyond that of current CMV based vectors may generally improve immunogenicity.
The art teaches that plasmid entry into the nucleus is a limiting factor in obtaining antigen expression. Increasing nuclear localization of a plasmid through inclusion of NFκB binding sites or a SV40 enhancer improves antigen expression in vitro and in vivo; this is presumed due to binding of NFκB which then piggybacks the plasmid to the nucleus (Dean D A, Dean B S, Muller S, Smith L C. 1999 Experimental Cell Research 253: 713-722). However, NFκB is generally localized in the cytoplasm, and transfer to the nucleus is limited, tissue-specific, and dependent on stimulatory signals. This limits the utility of NFκB nuclear targeting to improve DNA vaccination.
TH1 or TH2 bias: The art teaches that shifting immune response to DNA vaccine expressed viral or other antigens from TH2 to TH1 is desirable: to elevate humoral and cellular responses; and for other applications, such as allergy or instances where IgG1 (TH2) provide superior protection to IgG2a (TH1) a TH2 biased response is considered optimal. For example, CpG sequences (which promote TH1 response) improved antibody and cytotoxic T lymphocyte (CTL) responses to influenza HA, and CTL responses to influenza nucleoprotein DNA vaccines injected IM (Lee S W, Sung Y C. 1998 Immunology 94: 285-289). Co-immunization with IL12 or IL15 TH1 adjuvants improves T cell responses to influenza HA (Chattergoon M A, Saulino V, Shames J P, Stein J, Montaner L J, Weiner D B. 2004 Vaccine 22: 1744-1750; Kutzler M A, Robinson T M, Chattergoon M A, Choo D K, Choo A Y, Choe P Y, Ramamathan M P, Parkinson R, Kudchodkar S, Tamura Y, Sidhu M, Roopchand V, Kim J J, Pavlakis G N, Felber B K, Waldmann T A, Boyer J D, Weiner D B. 2005 J Immunol 175: 112-123) and antibody mediated protection (Operschall E, Pavlovic J, Nawrath M, Molling K. 2000 Intervirol 43: 322-330).
Immunostimulatory Adjuvants:
A number of microbial-specific motifs have been identified that activate innate immunity through Toll like receptor (TLR) binding, for example, Tri-acyl lipopeptides (TLR1/TLR2) peptidoglycan (TLR2), dsRNA (TLR3), bacterial HSP60 or Lipopolysaccharide (LPS; TLR4), flagellin (TLR5), Di-acyl lipopeptide (TLR6), ssRNA (TLR7, TLR8), unmethylated CpG DNA (TLR9). U-rich or U/G rich ssRNA TLR7/8 agonists have been identified that induce interferon responses (Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S. 2004 Science 303: 1526-1529; Diebold S S, Kaisho T, Hemmi H, Akira S, e Sousa C R. 2004 Science 303: 1529-1531; Barchet W, Krug A, Cella M, Newby C, Fischer J A A, Dzionek A, Pekosz A, Colonna M. 2005 Eur. J. Immunol. 35: 236-242) as well as a sequence specific siRNA that induces interferon production from human and mice plasmacytoid dendritic cells through TLR7 (Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S, Noronha A, Manoharan M, Akira S, de Fougerolles A, Endres S, Hartmann G. 2005 Nature Med. 11: 263-270). A novel class of immunostimulatory nucleic acid, single stranded CpG RNA which does not require TLR3, 7, 8 or 9 has also been identified (Sugiyama T, Gursel M, Takeshita F, Coban C, Conover J, Kaisho T, Akira S, Klinman D M, Ishii K J. 2005 J Immunol. 174: 2273-2279).
These molecules can be utilized as adjuvants to improve DNA vaccination through interferon production. However, exogenously applied adjuvant adds expense, complicates regulatory approval (an additional investigational entity in the vaccine) and requires high dosages since the adjuvant is not targeted (i.e. affects multiple cells in addition to cells containing the DNA vaccine); the high dose of untargeted adjuvant also presents special safety concerns (e.g. autoimmunity, inflammation, sepsis).
Unmethylated CpG is present in the vector backbone of microbial produced plasmids and augmentation (CpG enriched plasmids) can be used to stimulate TH1 responsive innate immune signals through TLR9. Unfortunately, these effects are observed only with high dosages, and CpG effects are minimal with advanced delivery methods which use economically low amounts of antigen (e.g. gene gun) as reflected by a TH2 biased response. As well, the overall poor immunological response to DNA vaccines in humans has been attributed, in part, to significantly reduced expression of TLR9 in humans compared to mice.
Vector encoded protein TLR agonists potentially would induce the innate immune system at low dose, since the signal from these elements is “amplified” from the vector (rather than a fixed vector component such as CpG). Incorporation of a flagellin producing gene into the vector backbone activates innate immune responses and potentiated TH1 bias and cellular immune response to an antigen delivered by Gene Gun. This demonstrates the potential for utilization of amplifiable TLR agonists to potentiate low dose DNA vaccination (Applequist S E, Rollman E, Wareing M D, Liden M, Rozell B, Hinkula J, Ljunggren H G. 2005 J Immunol. 175: 3882-3891). However, for inclusion of an innate immunity inducer in a DNA vaccine vector backbone, there should be no associated adaptive immune response since this would limit repeat usage and generate variable results in a population due to attenuated responses in individuals with prior exposure (preexisting immunity). Vectors such as alphaviral replicons (which produce dsRNA adjuvant) or the flagellin producing vector described above contain one or more proteins that can induce adaptive immunity to vector components and are unsuitable for repeat application.
Cell death: The art teaches that cell death can augment immune responses to antigens. IM injection of influenza HA and nucleoprotein (NP) DNA vaccines co-delivered with mutant caspases that promote slow cell death enhanced T cell responses and cellular immunity (Sasaki S, Amara R R, Oran A E, Smith J M, Robinson H L. 2001 Nature Biotechnol 19: 543-547). The immune response to influenza HA and NP is also dramatically enhanced (compared to DNA vaccines) utilizing Semliki forest alphavirus replicon (suicide) vaccines (Berglund P, Smerdou C, Fleeton M N, Tubulekas I, Liljestrom P. 1998 Nature Biotechnol 16: 562-565) that induce apoptosis; cell death is critical for the improved immune response. Replicon vectors contain multiple viral replication proteins; immune response against these proteins may limit repeat usage. Apoptotic cell death has also been accomplished by co-administering Fas or mutated caspases 2 or 3, which enhances CTL responses to intramuscularly (IM) administered DNA vaccines. Co-administering caspases also improves immune responses to influenza HA DNA vaccine by Gene Gun (Sasaki et al, Supra 2001). The optimal condition may be to selectively kill muscle or keratinocyte cells (but not immune cells) for a source of antigen for dendritic or langerhans cells (Reviewed in Leitner W W, Restifo N P. 2003 J Clin invest 112: 22-24). This is not possible utilizing constitutive cell death promoting agents Inhibition of apoptosis can also improve immune responses, wherein co-administering antiapoptotic Bcl-XL strongly enhanced T cell response after Gene Gun administration. This may reflect a benefit of prolonging dendritic cell lifespan. However, the use of cell death inhibitors may predispose cells to transformation (in the case of integrated plasmids) and increase cancer risk.
Cytoplasmic dsRNA activates PKR, ADAR, OAS, RIG-I and MDA5, which collectively induce interferon production, inhibit protein synthesis and edit or degrade RNA, thus reducing antigen production eventually leading to apoptotic cell death (reviewed in Wang Q, Carmichael G G. 2004 Microb. Molec. Biol. Rev. 68: 432-452). Cell death releases the dsRNA, which can then be taken up by cells, and further induce innate immune response by binding and stimulating endosomally localized TLR3 (Reviewed in Schroder M, Bowie A G. 2005 Trends Immunol. 26: 462-468). The art teaches that this type of dsRNA stimulation occurs with alphavirus replicon vaccines. Alphavirus replicon (suicide) vaccines induce enhanced immune responses with 100-1000 fold less antigen compared to standard DNA vaccines (by IM injection). These vectors induce apoptosis, presumed through formation of dsRNA which activates antiviral pathways and eventually leads to apoptotic cell death (Leitner W W, Ying H, Driver D A, Dubensky T W, Restifo N P. 2000 Cancer Research 60: 51-55). Cell death is required for improved vaccine efficacy and is mediated by cytoplasmic replicon dsRNA; it is possible that dsRNA in apoptotic elements are phagocytosed by APC's, and induce innate immunity through the endosomal TLR3 dsRNA recognition pathway. Co-delivery of anti-apoptotic gene (Bcl-XL) reduced protection, despite increasing antigen production (Leitner W W, Hwang L N, Bergmann-Leitner E S, Finkelstein S E, Frank S, Restifo N P. 2004 Vaccine 22: 1537-1544; Leitner W W, Hwang L N, DeVeer M J, Zhou A, Silverman R H, Williams B R G, Dubensky T W, Ying H, Restifo N P. 2003 Nature Med 9: 33-39; Matsumoto S, Miyagishi M, Akashi H, Nagai R, Taira K. 2005 J Biol Chem 280: 25687-25696). However, a delivery dependent balance between cell death signals and optimal production of antigen is required, since suicide DNA vaccines are not effective with Gene Gun delivery (which transfect dendritic cells) unless an anti-apoptosis gene is included (Kim T W, Hung C F, Juang J, He L, Hardwick J M, Wu T C. 2004 Gene Ther 11: 336-342).
Hone, D, Lewis, G, Fouts, T, Bagley, K, Boyson, M, Obriecht, C, Shata, M T, Agwale, S., 2003 World Patent Application WO0219968 disclose DNA vaccines that co-express antigens in combination with biologically-active components, such as adjuvants, immunoregulatory agents, antisense RNAs, and/or catalytic RNAs. Immunoregulatory agents are defined as peptides or proteins, and use of immunostimulatory RNA is not disclosed or contemplated by the inventors.
Pollo, J M, Dubensky, T W, Belli, B A, Perri, S, Fong, T. 2000 World Patent Application WO0061770 disclose expression cassettes comprising a promoter operably linked to a nucleic acid molecule which, when transcribed in vivo, forms double-stranded RNA (dsRNA) that induces the production of interferon. Compositions and methods were provided for the expression of noncoding dsRNA in the context of expression of a desired antigen with the object to enhance the overall robustness of antigen-specific immune responses. The authors teach that induction of Type I and II interferon's as a result of the intracellularly produced dsRNA in turn induces the synthesis of protein kinase R (PKR), and 2′-5 oligoadenylate synthetase (OAS), causing apoptosis and protein expression inhibition. A mechanism for induction of interferons by dsRNA is not disclosed. The authors do not teach activation of interferon production utilizing MDA5 or RIG-I signaling.
Williams J A 2006 World Patent Application WO2006078979 teach expression of immunostimulatory RNA from the vector may be used to activate cytoplasmic RNA pattern receptors such as PKR, RIG-I or MDA5, or, after cell death, TLR3 (dsRNA) or TLR7/8 (ssRNA) through uptake by bystander cells.
Even in view of the prior art, there remains a need for improved DNA vaccine vectors that are minimized to eliminate extraneous DNA, do not require antibiotic selection, are organized to ensure high quality bacterial plasmid productivity and improved in vivo expression, and induce improved innate and adaptive immune responses to the target antigen (but not the vector backbone), such that this technology can be utilized to meet the efficacy threshold in humans and other mammals, birds or fish with a wide range of target antigens.